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作 者:马凯歌[1] 邵增务[1] 王佰川[1] 熊蠡茗[1] 吴强[1] 杨述华[1]
机构地区:[1]华中科技大学同济医学院附属协和医院骨科,武汉430022
出 处:《中华实验外科杂志》2013年第2期354-357,共4页Chinese Journal of Experimental Surgery
基 金:国家自然科学基金资助项目(81171749、30872610、30700841)
摘 要:目的观察自噬是否参与压力诱导兔髓核细胞退变,探讨压力诱导髓核细胞退变机制。方法从3月龄兔胸腰段脊柱提取兔髓核细胞培养,取第2代兔髓核细胞,1Mpa压力环境下培养12、24、48h。细胞活力与细胞毒性检测观察压力对细胞生长的影响,透射电镜观察压力条件下细胞的微观结构。单丹磺酰尸胺(MDC)染色观察细胞内自噬性空泡结构及自噬率,实时荧光定量聚合酶链反应(FQ—PCR)观察自噬相关基因在压力条件下的表达。结果压力导致细胞衰老及死亡,透射电镜见细胞内存在自噬体结构,MDC染色后行流式细胞学检测显示12、24、48h自噬率分别为(1.580±0.171)%、(7.930±0.252)%、(13.530±1.206)%,PER结果显示压力诱导自噬相关基因(LC3B、Beclin-1)表达量与正常条件下表达明显增多(P〈0.05),且随着压力培养时间延长,LC3B、Beclin-1表达量也明显增多(P〈0.05)。结论自噬参与压力诱导细胞退变的过程,为压力诱导椎间盘髓核细胞退变的防治提供新的途径。Objective To investigate the involvement of autophagy in the progression of compres- sion-induced intervertebral disc (IVD) degeneration. Methods Rabbit nucleus pulposus (NP) cells were isolated from the thoracolumbar IVD of 3-month-old Japanese white rabbits. The rabbit NP cells at the sec- ond generation were cultured under the compression condition of 1 Mpa for 12, 24 or 48 h. Cell viability was determined by using cell counting Kit-8 (CCK8). The uhrastructural features of rabbit NP cells ex- posed to compression were examined under the transmission electron microscopy (TEM). The presence of autophagic vacuoles, as a marker of autophagy, was detected by using fluorescent dye monodansylcadaver- ine (MDC). The variation of autophagy-related gene expression was analyzed by using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) to evaluate the effect of compression on autophagy of rab- bit NP cells. Results Compression induced rabbit NP cell degeneration and death. The autophagosomes were detected in rabbit NP cells under the TEM. The autophagic rate was (1. 580 ± 0. 171 )% , (7. 930 ± 0. 252) %, ( 13. 530 ± 1. 206) % at 12, 24, and 48 h respectively. The mRNA expression of beclin-1 and LC3B was significantly higher in the compression treatment group than in the control group (P 〈 0. 05 ), and the mRNA expression of beclin-1 and LC3B was increased over time under compression culture condi- tion. Conclusion Autophagy and autophagic cell death were present in compression-induced rat NP cell injury, which offers a novel sight about compression-induced degeneration of IVD cells.
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